A variety of different mechanisms to combat pathogen challenges have emerged during metazoan evolution. The diverse nature of form, function and phylogenetic integration of these processes is significant for understanding basic mechanisms governing immune recognition. Acquisition of complete genome sequences in species that represent key points in phylogeny not only permit in-depth analysis of immune mediators but also represent an invaluable resource for understanding genetic mechanisms that create and maintain polymorphism and haplotype complexity. Such information is critical for gaining a more comprehensive view of: interrelationships within the innate immune system, the basis for complex polymorphisms in certain innate receptors and the origins of adaptive immunity. Amphioxus (a cephalochordate) occupies a unique phylogenetic position that represents the base of chordate evolution. Two striking features distinguish immune- related function in this species: 1) large-scale expansions and diversification have occurred in gene families encoding Toll-like receptor (TLR) innate immune receptors as well as other immune gene families and 2) a multigene family encoding immunoglobulin variable domain-containing chitin-binding proteins (VCBPs) exhibits an extraordinary level of regionalized hypervariation. In order to understand the genetics and functions of these phenomena, monomeric and multivalent, chimeric VCBP and TLR immune-type receptor genes will be engineered. In order to characterize ligands and ligand binding, conventional screening methods, metagenomic approaches and phage display will be adapted. Responses to immune stimulation will be characterized in cell types expressing VCBPs, TLR-related molecules and other innate receptors. Cellular specialization will be related to receptor diversity and mechanisms that sense pathogen signals. Transcriptional complexity of immune-related molecules will be addressed using a novel BAC transfection-transcriptome readout approach. Haplotypic variation will be characterized and germline as well as potential somatic variations will be examined. The mechanisms underlying the genetic variation seen in VCBPs at the natural population level will be examined by characterizing individual gametes. The relationships of genomic context, specifically repetitive DNA and noncoding haplotypic differences, to the germline complexity of VCBPs will be examined through direct study of meiotic variants as well as by plasmid- and BAC-transgenesis in zebrafish as a high throughput readout system. Collectively, a more comprehensive understanding of innate receptor diversification and function as well as relationships between polymorphism, functional variation and their relationship to chromosomal context will emerge. Such information is of particular relevance to current concepts of host responses to pathogen challenge and mechanisms used to create and maintain functional polymorphism of immune receptors.